1. Introduction
Fibroblast growth factor 2 (FGF2) is a member of the heparin-binding growth factor (HBGF) family, and is widely expressed in development and adult tissues (Nguyen et al. , 2013). FGF2 is a potent stimulator of cell proliferation, differentiation, and migration of multiple cell types, playing an essential role in embryonic development (Slacket al. , 1987), tissue repair (Maddaluno et al. , 2017), and angiogenesis (Chu et al. , 2011; Corseaux et al. , 2000). FGF2 can be used to accelerate the healing of both acute and chronic wounds. However, it has a short circulation half-life due to rapidly protease degradation, kidney filtration, and antigenic response, limiting its clinical application. Thus, increasing the stability of FGF2 is required to improve its application potential.
Although there are many chemical modification approaches to address stability, poly (ethylene) glycol (PEG) modification has been well demonstrated as an effective strategy to improve stability and biocompatibility of proteins (Brocchini et al. , 2008; DeFreeset al. , 2006; Krall et al. , 2016). PEG is a substance that has been designated by the Food and Drug Administration (FDA) as “Generally Recognized as Safe (GRAS)” (Parnaud et al. , 1999), and a variety of PEGylated proteins have been approved for use by FDA (Dozier et al. , 2015). The most widely used PEG modification method is to engineer a single cysteine into a protein, and then rapidly and quantitatively react this cysteine with a PEG-maleimide group, thus forming a protein-PEG conjugate (Foley et al. , 2007; Rosenet al. , 2017). Cysteine is an ideal target for site-specific protein modification due to its typical low abundance in proteins and the high nucleophilicity of the sulfhydryl side chain (Bernardimet al. , 2016). Previous efforts using site-selective PEGylation of cysteine residues have resulted in modified proteins with improved pharmacokinetics and retained biological activity (Dozier et al. , 2015).
The crystal structure of the FGF2-FGFR-Heparin ternary complex shows that FGF2 activity depends on the heparin-dependent formation of 2:2 FGF2-FGFR dimer complex (Beenken et al. , 2012). Heparin facilitates FGF-FGFR dimerization by binding both FGF and FGFR, and thereby promoting and stabilizing the protein-protein contacts between ligand and receptor (Beenken et al. , 2009). Obviously, the receptor binding region and heparin-binding region are important regions for the activity of FGF2. However, there has been little detailed study of structure-activity relationships in FGF2. Previous efforts to modify FGF2 focused on the protein’s N-terminus or chemical modification of two surface-exposed cysteines (Decker et al. , 2016; Kang et al. , 2010), but the resulting long-acting FGF2 conjugates exhibited reduced bioactivity. Therefore, a rational modification strategy based on the structure to select optimal sites on the protein for cysteine mutation may be more effective to obtain PEGylated proteins that retain biological activity.
To identify suitable modification sites, four surface-exposed sites, including two sites near the heparin- and FGFR-binding regions and two native cysteines were selected and substituted by cysteine or alanine, and PEG-FGF2 conjugates were synthesized and purified. Structure-activity analysis and long-acting characteristics of these conjugates were explored using in vitro and in vivo wound healing models.